Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4−CD8− double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

Systemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease of unknown etiology 1 .Its pathogenesis has been characterized by the production of antinuclear autoantibodies (ANA) with both T cells 2 and B cells being essential for disease development 3,4 .Activation of the mechanistic target of rapamycin (mTOR) within T cells was earlier uncovered as a therapeutic target in patients with systemic lupus erythematosus (SLE) 5,6 .Subsequently, mTOR was identified as a central regulator of lineage development in the immune system 7,8 .mTOR is considered to be an integrator of genetic and environmental cues, which confer predisposition to SLE [9][10][11] .In turn, blockade of mTOR with rapamycin has shown preliminary evidence for remarkable therapeutic efficacy both in mice [12][13][14] and patients with SLE 5,[15][16][17] .mTOR may be activated by oxidative stress 18,19 and localization to the lysosomal membranes where it senses amino acid sufficiency 20 .Skewed tryptophan metabolism with characteristic accumulation of kynurenine (KYN) has been identified as the top metabolic biomarker 21,22 and predictor of therapeutic mTOR blockade in patient with SLE 21 .KYN triggered the activation of mTORC1 in primary T cells of patients 21 and mice with SLE 23,24 .Metabolome analyses demonstrated that lupus T cells processed tryptophan (TRP) differently, suggesting a contribution of T-cell intrinsic factors 24 .However, the precise mechanism of KYN-sensitive mTOR activation during T-cell development and lupus pathogenesis has been unknown.mTOR has been localized to endosomes 25 along with traffic regulator small GTPases, Rab4A 6 and Rab5 26 .Both of these GTPases are overexpressed in T cells of patients 6 and mice with SLE 13 .Notably, the overexpression of Rab4A, but not Rab5, precedes mTOR activation, ANA production and disease onset in SLE 13 .Rab4A is encoded by the HRES-1/Rab4 human endogenous retroviral element 27 , which is centrally positioned within the 1q42 lupus susceptibility locus 28 .Endogenous retroviruses, such as HRES-1/Rab4, share regulatory DNA elements with exogenous viruses and serve as sensors of infections 27 and mediators of autoimmunity 29,30 .Polymorphic alleles of HRES-1/ Rab4 have been associated with autoantibody production and predisposition to autoimmune diseases, such as SLE 31,32 and multiple sclerosis (MS) 33,34 .HRES-1 haplotypes influence autoantibody production and organ involvement, including glomerulonephritis (GN), in patients with SLE 32 .HRES-1 polymorphisms influence the expression of HRES-1/Rab4 or Rab4A 27,35 , a small GTPase that regulates endosomal recycling of surface receptors and organelles, including mitochondria 36 .Rab4A restrains mitophagy and promotes the accumulation of oxidative-stress-generating mitochondria 13,37 .Further upstream, the transcription of Rab4A is controlled by redox-sensitive transcription factors, NRF1 and USF1 35 .Downstream, Rab4A itself regulates mTOR activation in Jurkat human leukemic T cells and primary human T lymphocytes in vitro 35 .However, the role of Rab4Amediated endosome traffic beyond mTOR activation, T-cell lineage development and autoimmunity remain unknown.
Here, we show that constitutive activation of Rab4A in C57Bl/6 (B6) and lupus-prone B6 SLE1.2.3.triple-congenic (B6.TC) mice exert dominant control over pro-inflammatory signal transduction networks at multiple levels in vivo: i) Rab4A enhances mitochondrial metabolism by triggering the accumulation of mitochondria, mitochondrial hyperpolarization (MHP), increased mitochondrial ATP production and enhanced tricarboxylic acid (TCA) and pentose phosphate pathway (PPP) fluxing in CD4 + T cells; ii) Rab4A prominently accelerates the recycling and surface expression of CD98 that serves as receptor for branched chain and aromatic amino acids and pro-inflammatory metabolites, such as KYN.In turn, KYN may spread inflammation through the bloodstream by eliciting mTOR activation, the expansion of CD4 + T cells at the expense of CD8 + T cells, promotes CD3 + CD4 − CD8 − double-negative (DN) T cell and B cell activation and plasma cell expansion; iii) Rab4A-driven mTOR activation promotes ANA production and GN, while the inactivation of Rab4A in T cells restrains CD98 expression, KYN accumulation, mTOR activation, mitochondrial metabolism and lineage skewing and blocks ANA and antiphospholipid antibody (aPL) production and GN.Moreover, CD98 expression is elevated in T cells and it predicts therapeutic response to mTOR blockade within the context of a controlled clinical trial in patients with SLE 15 .These results establish Rab4A as a cell type-specific controller of mitochondrial metabolism and CD98-dependent and KYN-sensitive mTOR activation that mediate therapeutically targetable pro-inflammatory lineage specification in SLE.

Results
Activation of Rab4A promotes autoimmunity and glomerulonephritis in female lupus-prone B6.TC mice Rab4A is overexpressed in T cells of SLE patients 6 and, prior to the onset of ANA production or any sign of disease, in lupus-prone mice 13 .
To determine its causative involvement in disease pathogenesis, we replaced the wild-type Rab4A alleles in C57Bl/6 (B6) and lupus-prone B6 SLE123 triple congenic (B6.TC) mice with constitutively active Rab4A Q72L alleles surrounded by loxP sites (B6/Rab4A Q72L ; Figures S1A,  B, and C).Rab4A was selectively deleted in T cells of B6/Rab4A Q72L -KO and B6.TC/Rab4A Q72L -KO mice by crossing of B6/Rab4A Q72L and B6.TC/ Rab4A Q72L strains with B6.CD4 Cre mice 38 (Figures S1D and E).Cre was not expressed in peripheral CD8 + T cells as the deletion of Rab4A had occurred at the developmental stage of double-positive T cells in the thymus, as originally described in this targeted deletion model 38 .The production of ANA marked the onset of autoimmunity in male and female B6.TC mice from 20-29 weeks of age (Fig. 1A).Constitutive Rab4A activation enhanced ANA production in both female and male B6.TC/Rab4A Q72L mice over B6.TC controls matched for age and gender (Fig. 1A).Interestingly, ANA production was also increased B6/ Rab4A Q72L over B6 controls, suggesting that activation of Rab4A alone also triggers autoimmunity on the B6 background (Fig. 1A).Inactivation of Rab4A in T cells blocked ANA production in female but not male B6.TC/Rab4A Q72L -KO mice over B6.TC/Rab4A Q72L controls (Fig. 1A).
Kidney tissues were scored for GN, glomerulosclerosis (GS), and % of glomeruli with sclerosis or hyalinosis at 50 weeks of age by an expert renal pathologist who was blinded to genotypes or therapeutic interventions, as earlier described 13 .Both female (Fig. 1E) and male lupusprone B6.TC mice had significant increases in GN scores relative to B6 controls at 50 weeks of age (Fig. 1F).As expected 42 , females had more severe GN scores (1.31 ± 0.23) than male B6.TC mice (0.61 ± 0.19; p = 0.0282).Importantly, female B6.TC/Rab4A Q72L lupus-prone mice carrying constitutively active Rab4A Q72L alleles had more severe GN than B6.TC lupus-prone mice with wild-type Rab4A alleles (Fig. 1E; p = 0.0295).By contrast, inactivation of Rab4A in T cells completely abrogated GN in B6.TC/Rab4A Q72L -KO female mice relative to B6.TC/ Rab4A Q72L controls (Fig. 1E; p < 0.0001).GN scores of B6.TC/Rab4A Q72L -KO mice were also reduced relative to B6.TC controls (Fig. 1E; p = 0.0045).While Rab4A activation triggered autoimmunity both in females and males (Fig. 1A-C), it failed to influence proteinuria and GN in male B6.TC mice (Fig. 1F), which may be attributed to gender differences in end-organ resistance in SLE 43 .Interestingly, male B6.TC/ Rab4A Q72L -KO mice developed severe glomerulosclerosis with greater percentage of glomeruli with sclerosis or hyalinosis relative to B6.TC mice with normal Rab4A alleles (Fig. 1F).These findings are consistent with earlier observations that males have elevated glomerulosclerosis index, mean glomerular volume, and proteinuria (3.1-, 1.7-, and 1.8-fold, respectively) over age-matched females 40 .Importantly, estrogen regulates the biosynthesis of geranylgeranyl isoprene units which allow for posttranslational modification of Rab GTPases, such as Rab4 44 .While geranylgeranylation is required for binding of Rab4A to endosome membranes, pharmacological blockade of this enzymatic process inhibits the development of SLE in female mice 13 .Therefore, Rab4A-directed therapy may have different outcomes in males relative to females.SLE has a 9:1 increased prevalence in female over male patients with GN being a leading cause of mortality 45 .Given the overexpression of Rab4A in SLE patients, predisposition to GN in female B6.TC/Rab4A Q72L mice, and blockade of GN upon inactivation of Rab4A in T cells, its role in immune system activation and disease pathogenesis were further investigated in female mice.
Rab4A expands CD4 + T cells at the expense of CD8 + T cells both in B6 and B6.TC mice In order to determine the mechanisms by which Rab4A promotes the immunopathogenesis of SLE, lymphocyte subsets were examined in female mice before the onset of autoantibody production and proteinuria at 20 weeks of age.T cells were overall expanded at the expense of B cells in the spleen of B6.TC/Rab4A Q72L mice over B6/ Rab4A Q72L controls (2-way ANOVA p < 0.0001; Fig. 2A).Within T cells, Rab4A activation expanded CD4 + T cells but depleted CD8 + T cells both in B6/Rab4A Q72L and B6.TC/Rab4A Q72L mice over B6 and B6.TC controls, respectively (Fig. 2B).By contrast, CD4 + T cells were depleted in Rab4A Q72L -KO mice with respect to Rab4A Q72L parental controls both in the B6 and B6.TC strains, reversing the CD4:CD8 ratio back to baseline.These findings suggest that the activation of Rab4A causes an expansion of CD4 + cells at the expense of CD8 + cells within the T cell compartment in both B6 and B6.TC mice.The inactivation of Rab4A in T cells of B6/Rab4A Q72L -KO and B6.TC/Rab4A Q72L -KO mice contracted the relative and absolute numbers of CD4 + T cells in comparison to B6/ Rab4A Q72L and B6.TC/Rab4A Q72L mice, respectively (Figs. 2B, S3).In contrast, relative rather than absolute numbers of CD8 + T cells were expanded by the inactivation of Rab4A in T cells of B6.TC/ Rab4A Q72L -KO mice over B6.TC/Rab4A Q72L controls (Figs.2B, S3).These findings indicate that the skewing of CD4:CD8 T-cell abundance by Rab4A may be driven by the absolute depletion of CD4 + T cells rather than the expansion of CD8 + T cells in B6.TC/Rab4A Q72L -KO mice over B6.TC and B6.TC/Rab4A Q72L controls (Fig. S3).
In accordance with earlier findings that Rab4A limited mitophagy and promoted the accumulation of mitochondria in HeLa and Jurkat cells 13,37 , the metabolic basis of lineage skewing was characterized by an increase of mitochondrial mass in CD4 + T cells of B6.TC/Rab4A Q72L mice over B6.TC controls, which were reversed upon Rab4A deletion in B6.TC/Rab4A Q72L -KO mice (Fig. 2C).Relative to the mitochondrial mass, the mitochondrial transmembrane potential (ΔΨm) was elevated in CD4 + T cells of B6.TC/Rab4A Q72L mice, indicating MHP (Fig. 2C), which is a hallmark of mitochondrial dysfunction of T cells of patients with SLE 46 .Activation of Rab4A failed to augment mitochondrial mass or elicited MHP in CD8 + T cells of B6/Rab4A Q72L mice (Fig. 2D).
Rab4A enhances glucose flux and R5P production through the PPP in CD4 + but not in CD8 + T cells Unlike mitochondrial respiration, glycolysis was unaffected by Rab4A activation alone in CD4 + or CD8 + T cells when measured with the Seahorse metabolic analyzer (Fig. S12A).However, in vivo treatment with rapamycin imposed markedly opposite effects on glycolysis of CD4 + and CD8 + T cells, which were influenced by Rab4A (Fig. S12A).Upon rapamycin treatment alone, both glycolysis and glycolytic capacity were enhanced in CD4 + T cells but reduced in CD8 + T cells of B6.TC mice (Fig. S12B).Rapamycin also increased glycolysis and glycolytic capacity in CD4 + T cells of B6.TC/Rab4A Q72L -KO mice (Fig. S12A).Following rapamycin treatment in vivo, glycolysis and glycolytic capacity were enhanced in CD8 + T cells of B6.TC/Rab4A Q72L mice relative to those from B6.TC controls (ANOVA p = 0.0004; Fig. S12A).Thus, Rab4A exerted cell type-specific changes in glycolysis between CD4 + and CD8 + T cells in the setting of mTOR blockade.

KYN induces CD4 + T-cell expansion, CD8 + T-cell depletion, B-cell activation, and plasma cell expansion amongst mouse splenocytes
Among the metabolites accumulated in the sera of B6.TC/Rab4A Q72L mice (Fig. 5A), KYN in and of itself elicited the accumulation of mitochondria and the production of reactive oxygen intermediates (ROS) (Fig. 6A) and increased CD98 expression in CD4 + and CD8 + T cells (Fig. 6B).As measured by mean fluorescence intensity (MFI), the extent of CD98 expression was greater on CD8 + T cells (ANOVA p < 0.0001) that occurred with the contraction CD8 + T cells relative to CD4 + T cells upon treatment with KYN and concurrent CD3/CD28 co-stimulation (ANOVA p = 0.0059; Fig. 6B).The cell type-specific differences in KYN accumulation and KYN-induced contraction of CD8 + T cells may be attributed to markedly elevated expression of CD98 on CD8 + T cells over CD4 + T cells upon CD3/CD28 co-stimulation (ANOVA p = 0.0100; Fig. 6B).KYN increased mTORC1 and mTORC2 activity both in CD4 + (Fig. 6C) and CD8 + T cells (Fig. 6D).

CD98 controls KYN uptake, TCA metabolism, and redox homeostasis in HeLa cells
CD98 is a ubiquitously expressed surface protein 75 that mediates the transport of branched-chain (VAL, LEU, ILE) and aromatic amino acids (PHE, TYR, TRP) 76 , and therefore, we examined the effects of its siRNAmediated knockdown on these metabolic pathways and signal transduction in HeLa cells.CD98, also called SLC3A2, is a disulfide-linked heterodimer composed of a glycosylated heavy chain and a nonglycosylated light chain, large amino acid transporter 1 (LAT1), also called SLC7A5 77 .CD98 is detected as a 70-125 kD protein depending on the extent of glycosylation.CD98 has been localized to the cell surface 64 , endosomes and lysosomes 63 , where it has been implicated in activating mTORC1 78 .As shown in Fig. S14A, the selective knockdown of CD98, but not LAT1, reduced Akt phosphorylation (Fig. S14B) and distorted metabolic pathways led by changes in branched-chain and aromatic amino acid biosynthesis and degradation as well as the TCA cycle (Fig. S14C, D).LAT1 protein levels were not affected by Rab4A in CD4 + or CD8 + T cells of age-matched female B6.TC, B6.TC/Rab4A Q72L , and B6.TC/Rab4A Q72L -KO mice (Fig. S15A) or in Jurkat human CD4 + T cell lines with altered expression of Rab4A (Fig. S15B).However, CD98 knockdown markedly reduced intracellular levels of branched-chain (VAL, LEU, ILE) and aromatic amino acids (PHE, TYR, TRP) as well as well as KYN.Moreover, CD98 knockdown exerted antioxidant effects, as evidenced by increased GSH/GSSG ratio and reduced homocysteine levels (Fig. S14F).While KYN is a driver of mitochondrial oxidative stress 79 , its metabolism is dependent on the availability of a TCA metabolite, αKG [80][81][82][83] .Accordingly, the depletion of KYN was accompanied by the accumulation of αKG and other TCA metabolites upon CD98 knockdown (Fig. S14G).These findings suggest that CD98 expression impacts metabolic cross-talk between KYN metabolism and the TCA cycle.Rab4A forms a positive feed-back loop with CD98 and mTOR in patients with SLE Similar to lupus-prone B6.TC/Rab4A Q72L mice, flow cytometry of SLE and control participants, who had been matched for age and gender in the context of a clinical trial 15 , unveiled an expansion of CD98 + DN T cells in SLE patients (Fig. 9A, B). mTORC1 activation was confined to CD98 + T cells in SLE patients (Fig. 9B).Knockdown of CD98 by siRNA blocked CD3/CD28-induced expression of CD98 and restrained the activation of mTORC1 in primary human T cells (Fig. 9C).Overexpression of Rab4A enhanced the total protein levels (Fig. 9D) and increased the surface expression of CD98 in Jurkat cells (Fig. 9E).Thus, Rab4A-induced overexpression of CD98 may contribute to the  activation of mTORC1 in SLE.Similar to lupus T cells 6 , FKBP12 expression was also induced by Rab4A in Jurkat cells (Fig. 9D).Further, elevated expression of CD98 expression was predictive of therapeutic efficacy of sirolimus, as measured by the SLE responder index (Fig. 9F).
As earlier uncovered, expression of Rab4A was enhanced by activation of mTORC1 6 and, reciprocally, Rab4A also promoted mTORC1 activation in Jurkat and primary human T cells 35 .Of note, the overexpression of wild-type Rab4A enhanced mTOR localization to the lysosomes, whereas dominant-negative Rab4A S27N inhibited mTOR traffic to the lysosomes in Jurkat cells (Fig. S16A, B). mTOR activation depends on its localization to the lysosomal membranes where it senses amino acid sufficiency 20 .These findings thus suggest that enhanced traffic to the lysosome underlies Rab4A-dependent mTOR activation in Jurkat cells (Fig. S16C).

Rab4A accelerates MOG-induced EAE
Since HRES-1/Rab4 polymorphism has also been linked to MS 33,84 , we examined the impact of Rab4A in experimental autoimmune encephalitis (EAE), a mouse model for MS that is mediated by antigenspecific CD4 + T cells 85 .MOG-induced EAE was markedly enhanced in 12week-old B6/Rab4A Q72L mice over B6/WT controls, which was reversed upon deletion of Rab4A in T cells in B6/Rab4A Q72L -KO mice (Fig. S17).Enhanced EAE was characterized greater lymphocytic infiltration as well as vasculitis, resulting in hemorrhage of the spinal cord in B6/ Rab4A Q72L mice (Fig. S17).
through the TCA cycle.These CD8 + T cells exhibited oxidative stress characterized by the accumulation of MDA, and the depletion of NADH, which is required for activity of ETC complex I (Fig. 5).Rab4A activation in CD8 + T cells increased the production of pyridine nucleotide precursors, such as KYN, 3OH-KYN, QUIN, and nicotinate.Moreover, independent lines of evidence support a pro-inflammatory role for KYN: 1) the accumulation of KYN in sera and CD8 + T cells preceded the onset of autoantibody production and GN disease onset in SLE in B6.TC/ Rab4A Q72L mice; 2) KYN itself enhanced CD4 + over CD8 + T-cell development in primary B6 mouse splenocytes; 3) KYN activated mTOR in B cells and expanded plasma cells in primary B6 mouse splenocytes; and 4) Rab4A formed a positive feed-back loop with mTOR activation and expression of metabolite-transporting CD98 receptor during lupus pathogenesis in mice and patients with SLE.Furthermore, KYN → KYNA accumulation occurred with the depletion of αKG in CD8 + but not CD4 + T cells of B6.TC/Rab4A Q72L mice, indicating a Rab4A-driven cell typespecific crosstalk between KYN metabolism and the mitochondrial TCA cycle (Figs.5, S19).These findings identify CD98-dependent accumulation of KYN, as a pro-inflammatory metabolite that may contribute to Rab4A/mTOR-driven autoimmunity in SLE.
As also unveiled by this study, Rab4A elicited mTOR activation in CD4 + and CD8 + T cells of B6/Rab4A Q72L and B6.TC/Rab4A Q72L mice over involvement in TCR-induced mTOR activation.Alexa647-conjugated CD98 or scrambled control siRNA was electroporated into 2 × 10 6 HC peripheral blood lymphocytes (PBL), as earlier described 6,35 .CD98PE and pS6RP-AF488 histograms were gated on Alexa 647 + /CD3-APC-Cy7 + dual-positive cells.mTORC1 activation was assessed by the bracketed pS6RP-AF488 + cells, as earlier described 19,102 .D Rab4A promotes the surface expression of CD98.The effect of Rab4A was examined by western blot on the expression of FKBP12 and CD98 in Jurkat cells carrying doxycycline-inducible adeno-associated virus (AAV) expression vectors 27 .Western blots represent 5 or more similar experiments.Jurkat cells with construct 6678 overexpressed wild-type Rab4A while those with construct 9035 overexpressed dominant-negative Rab4A S26N (Rab4A-DN), as earlier described 27 .Control cells carried "empty" vector construct 4480 27 .Rab4A, FKBP12, CD4, and actin were detected by antibodies described earlier 6 .CD98 was detected with antibody sc-9160 from Santa Cruz Biotechnology (Santa Cruz, CA).E Effect of Rab4A on surface expression of CD98 was detected by flow cytometry.CD4 was detected as a control antigen that is targeted for lysosomal degradation by Rab4A, which is blocked by overexpression of Rab4A-DN 27 .Of note, the expression vectors confer moderate overexpression of Rab4A and Rab4A-DN in the absence of doxycycline, which are sufficient to exert opposing changes on CD4 expression in Jurkat cells of primary CD4 T cells, as earlier described 27 .Top and middle panels show histogram and dot plot overlays of CD4 and CD98 expression.Bottom panels show mean channel fluorescence intensity (MFI) of representative histograms of the top panel, while bar charts show mean ± SE of three independent experiments.P values represent comparison using two-tailed paired t test, connecting bars indicate P < 0.05, which reflect hypothesis testing and have not been corrected for multiple comparisons.F Effect of sirolimus (rapamycin) treatment on expression of CD98 in DN T cells in SLE patients during 12-month intervention.The prevalence of CD98 + DN cells was determined in thirteen freshly isolated PBL of SLE and HC participants matched for age within 10 years (top panel).Nine patients met criteria for SLE Responder Index (SRI + , middle panel), while 4 patients were SRI non-responders (SRI − , lowest panel).CD98 + DN cells were assessed before treatment (visit 1) and after treatment for 1 month (visit 2), 3 months (visit 3), 6 months (visit 4), 9 months (visit 5), and 12 months (visit 6).Effects of sirolimus were also assessed by 2-tailed paired t test relative to HC participants tested in parallel; *, p < 0.05.Charts show mean ± SE of patients and controls for each time point.
B6 and B6.TC controls which was consistently reversed upon the inactivation of Rab4A in T cells of B6/Rab4A Q72L -KO and B6.TC/ Rab4A Q72L -KO mice.Prior to the onset of autoimmunity, mTORC1 was activated in CD4 + and CD8 + T cells of 20-week-old B6.TC/Rab4A Q72L mice relative to B6/Rab4A Q72L controls.This suggests that Rab4Amediated mTOR activation is a driver of autoimmunity via expansion of CD4 + over CD8 + T cells in SLE.A positive feed-back loop between Rab4A and mTOR was further substantiated by the reversal of mitochondrial changes in CD4 + and CD8 + T cells of rapamycin-treated B6.TC/Rab4A Q72L mice.In mediating such fundamental control of T cell lineage development, Rab4A markedly distorted gene expression into sharply opposite directions between CD4 + T cells and CD8 + T cells, primarily affecting mitochondrial metabolism, endosome traffic, and autophagy pathways.The differential effect by rapamycin on metabolic flux between CD4 + and CD8 + T cells may be attributed, at least in part, to the variable reliance of these cells on glycolysis 86 relative to the mitochondrial TCA cycle 87 , respectively.
The accumulation of mitochondria in CD4 + T cells of B6/Rab4A Q72L and B6.TC/Rab4A Q72L mice is consistent with a role for Rab4A in depleting Drp1 and thus limiting mitophagy in lupus T cells 13,37 .Rab4Amediated depletion of Drp1 was found to be mTOR dependent both in vitro and in vivo, as the retention of mitochondria in T cells of B6.TC/ Rab4A Q72L mice was reversed by rapamycin treatment.The accumulation of oxidative stress-generating mitochondria in lupus T cells 13,46 has been widely confirmed 88,89 and recently extended to myeloid cells, such as neutrophils 90,91 and erythroid cells of patients with SLE 92 .While mitochondrial dysfunction of erythroid cells was also attributed to defective mitophagy, the involvement of Rab4A and mTOR activation have not been addressed in this system 92 .
Beyond trafficking organelles and intracellular proteins to lysosomes 93 , a systematic analysis of surface receptors that recycle via endosomes unveiled a mosaic of Rab4A dependency.Canonical PDBuinduced internalization 27,94 and subsequent recycling and expression CD71 were enhanced by Rab4A in CD4 + T cells of B6.TC/Rab4A Q72L mice (Fig. S18).Most recently, overexpression of CD71 was associated with enhanced iron uptake into CD4 + lupus T cells 95 , which might contribute to mitochondrial dysfunction in SLE independent of mTOR pathway activation.As unveiled by this study, the surface expression and traffic of CD98 followed a different pattern, as it failed to internalize after PDBu treatment, and lupus itself promoted the recycling and expression of CD98 in B6.TC mice as compared to non-autoimmune B6 controls (Fig. S18).Expression of CD98 was synergistically promoted by Rab4A activation and SLE on all T cells of B6.TC/Rab4A Q72L mice; these coordinate changes were consistently reversed by the inactivation of Rab4A in B6.TC/Rab4A Q72L -KO mice.Increased expression of CD98 was mechanistically connected to a robust positive feed-back loop within a Rab4A-mTOR-CD98 axis in mice and patients with SLE.While Rab4A promoted mTOR activation and CD98 expression, mTOR blockade reduced the expression of Rab4A and CD98.Furthermore, knockdown of CD98 in primary human T cells blocked CD3/CD28induced activation of mTORC1, suggesting that CD98 transmits critical signals required for mTOR activation and T-cell function.As revealed by this study, CD98 supports the uptake of branched-chain (VAL, LEU, ILE) and aromatic amino acids, TRP and its metabolite KYN.While KYN itself activated mTORC1 and mTORC2 in CD4 + and CD8 + T cells, it preferentially stimulated CD98 expression and depletion of CD8 + T cells.Recently, KYN uptake was attributed to SLC7A5 (LAT1) and associated with mTORC1 activation in dendritic cells in SLE 96 .Therefore, it is conceivable that both the CD98 heavy chain (SLC3A2) and the LAT1 light chain (SLC7A5) of the heterodimer can independently regulate KYN uptake.These findings support earlier observations that the accumulation of KYN may contribute to mTOR activation in patients with SLE 21 .mTORC1 and mTORC2 were activated in CD19 + , CD19 + CD38 + , and CD19 + CD11c + ABCs of B6.TC/Rab4A Q72L mice.These changes were all reversed by the inactivation of Rab4A in T cells of B6.TC/Rab4A Q72L -KO mice.Rapamycin and NAC also reversed the activation of mTORC1 and mTORC2 and the expansion of ABCs in B6.TC/Rab4A Q72L mice.Of note, elevated production of ANA and aPL occurred with the accumulation of KYN in sera of B6.TC/Rab4A Q72L mice.KYN activated mTORC1 and mTORC2 and augmented the expression of CD98 in CD19 + , CD19 + CD38 + B cells, CD19 + CD11c + ABCs, and CD138 + plasma cells.Moreover, KYN also expanded plasma cells with concurrent LPS stimulation.These findings suggest that population shifts in the B-cell compartment are driven by the accumulation of pro-inflammatory KYN metabolites in sera of B6.TC/Rab4A Q72L mice.
The overexpression of Rab4A preceded mTOR activation in several spontaneously lupus-prone mouse strains, including B6.TC, NZB/ WF1, MRL, lpr, and MRL/lpr mice 13 .DN T cells, preceding the later stages of CD4 + CD8 + DP and CD4 + or CD8 + SP T-cell development in the thymus, were depleted in B6/Rab4A Q72L females but expanded in B6.TC/Rab4A Q72L females along with coordinate skewing in activation of mTORC1 and mTORC2.These results suggest that Rab4A and lupusdriven inflammation exert distinct influences of T-cell development that warrant further studies.It has been firmly established that DN T cells may derive from activated CD8 + T cells in patients 97 and mice with SLE 98,99 .Given that the expansion of DN T cells is mTORdependent 15,100-102 , Rab4A-mediated mTOR activation may contribute to the depletion of CD8 + T cells and reciprocal expansion of DN T cells in B6.TC/Rab4A Q72L mice.This mechanism is supported by the effective reversal of DN T-cell expansion in rapamycin-treated B6.TC/Rab4A Q72L mice.Our results also indicates the heterogeneity of DN T cells that have been considered both drivers 103 and inhibitors of renal inflammation 104 .1) DN T cells were depleted in 20-week-old B6.TC/ Rab4A Q72L mice before the onset of SLE, as shown in Fig. 2; however, DN T cells were expanded in B6.TC/Rab4A Q72L mice and depleted in B6.TC/ Rab4A Q72L -KO mice after the onset of SLE. 2) While rapamycin restrained DN T cells in B6.TC/Rab4A Q72L mice with therapeutic efficacy, DN T cells were expanded in rapamycin-treated B6.TC/Rab4A Q72L -KO mice over rapamycin-treated B6.TC/Rab4A Q72L mice.These results reveal that the abundance of DN T cells is controlled by two different mechanisms in SLE: i) expansion via Rab4A-dependent mTOR activation as noted in B6.TC/Rab4A Q72L mice; and ii) contraction via Rab4Aindependent mTOR activation in B6.TC/Rab4A Q72L -KO mice, both of which can be reversed by treatment with rapamycin.Therefore, these two types of DN T cells may play divergent roles in disease pathogenesis 103,104 , which warrant further investigations.
Importantly, this study identifies Rab4A as a promoter of mTOR activation, ANA production, proteinuria, and GN during lupus pathogenesis in vivo.mTOR blockade by rapamycin or NAC reduced GN of B6.TC/Rab4A Q72L mice.These findings are consistent with the promising efficacy of mTOR blockade in SLE patients 15,19 , including those with GN 16,17,56,105 .Similar to SLE patients 15 , rapamycin blocked thrombocytopenia in B6.TC and B6.TC/Rab4A Q72L mice.Rapamycin increased hemoglobin in B6.TC/Rab4A Q72L and B6.TC/Rab4A Q72L -KO mice, suggesting that mTOR activation contributed to anemia in SLE.NAC blocked lupus GN and thrombocytopenia in B6.TC/Rab4A Q72L mice, which have not yet been observed in human participants 19 .
C alleles of the rs451401 single nucleotide polymorphism (SNP) in the HRES-1/Rab4 human genomic locus predispose to anti-DNA and aPL production and GN in patients with SLE 32 .This SNP has also been linked to another autoimmune disease, MS 33,84 .The predisposition of B6/Rab4A Q72L mice to MOG-induced EAE indicates that Rab4A activation may broadly enhance CD4 + T cell-dependent autoimmune diseases, including MS 106 .Another MS susceptibility gene 107 , C-type lectinlike domain family 16 member A, CLEC16A, has been recently mapped to Rab4A + recycling endosome in human T cells 108 .rs451401 C alleles of the transcriptional enhancer facilitate the expression of Rab4A and mTOR activation both in healthy and SLE participants 35 .As documented in this study, mTOR is trafficked by Rab4A to the lysosome, where it gets activated within the cell 109,110 .However, mTOR activation and downstream lineage development also depend on Rab4A-directed traffic of surface receptors, such as CD98.In CD4 + , CD8 + , and DN T cells, Rab4A promoted the recycling of CD98 that transports KYN which activates mTOR.Along this line, Rab4A and mTOR-dependent production of KYN is identified as pro-inflammatory metabolite that may transmit activation signals from CD8 + T cells to CD19 + and CD19 + CD38 + B cells and CD138 + plasma cells.Thus, signal transduction along the Rab4A-CD98-KYN-mTOR axis is hereby established as a positive feed-back loop underlying pro-inflammatory lineage specification in the immune system with considerable impact on the development of SLE.The contribution of this axis is likely to be central for disease pathogenesis and offer regulatory checkpoints as targets for therapeutic interventions in autoimmune disease beyond SLE.
Overexpression of Rab4A has been detected in T cells of SLE patients as well as T cells, B cells, macrophages, thymocytes and hepatocytes in multiple lupus-prone mouse strains 13,111 .Therefore, we created B6/Rab4A Q72L and B6.TC/Rab4A Q72L mice to examine the global impact of Rab4A activation both in B6 control and lupus-prone B6.TC mice.Accelerated lupus in B6.TC/Rab4A Q72L mice is consistent with the notion that the genetic polymorphism, which influences lupus susceptibility 35 , operates via activation of Rab4A.In accordance with the prominence of Rab4A overexpression in CD4 + T cells of SLE patients 6 , autoimmunity, lupus GN and EAE were all blocked in B6.TC/ Rab4A Q72L -KO mice lacking Rab4A in T cells.Since CD4 is expressed during the CD4 + CD8 + double-positive stage of T-cell development 38 , Rab4A was deleted both in CD4 + and CD8 + T cells.Although the results unveil a critical role of Rab4A in T cell development that controls the abundance of CD4 + over CD8 + T cells, its involvement in other relevant types of cells within and outside the immune system has not been evaluated 91,112 .In vivo and in vitro mechanistic studies identified the Rab4A-mTOR-CD98 positive feedback loop as a driver of proinflammatory lineage specification both in patients and mice with SLE.However, we have not individually targeted other trafficked receptors for impact on mitochondrial metabolism, mTOR activation, and disease pathogenesis.We identified increased production of KYN as a central metabolite that may spread mTOR activation and inflammation through the bloodstream.Although KYN accumulation in CD8 + T cells and sera of B6.TC/Rab4A Q72L mice occur with increased demand for pyridine nucleotides during lupus pathogenesis, all of which are mTOR-dependent, further mechanistic studies that connect KYN metabolism with mitochondrial dysfunction and metabolic flux through the TCA cycle seem warranted.These findings and limitations open up unexplored avenues for investigating metabolic control of immune cell development and the pathogenesis of autoimmunity.

Methods
These studies were conducted in compliance with all ethical regulations and study protocols approved by Institutional Review Boards of the State University of New York Upstate Medical University.The biomarker studies of human participants were conducted in the setting of a clinical trial (NCT00779194) 15 .

Mice
Autoimmunity-resistant C57BL/6 (B6) and lupus-prone B6.Sle1.2.3 triple congenic (B6.TC) mice 113 were obtained from Jackson Laboratory (Bar Harbor, ME).Since Rab4A is overexpressed in T cells of SLE patients 6 as well as T and B cells of MRL, lpr, NZB/W(F1), and B6.TC mice prior to the onset of ANA production or any sign of disease, we created mice with constitutively active Rab4A.We replaced exons 3 of the Rab4A genomic lupus in 129SvJ-derived TC1 embryonic stem (ES) cells using a targeting vector with 5 kb upstream and 3 kb downstream genomic fragments harboring exon 2 and exons 4 and 5 respectively, of the genomic locus (Figure S1).Intron 3 contained a neomycin resistance cassette flanked by Frt recognition motifs.Intron 2 and intron 3 downstream of the neomycin cassette harbored LoxP sites to allow the removal of exon 3 by crossing with transgenic mice expressing Cre recombinase (Figure S1A).Exon 3 was altered via site-directed A → T mutagenesis of codon CAG to CTG thus replacing amino acid Q 72 with L 72 (Fig. S1B, C).The targeting vector was electroporated into 129SvJ-derived TC1 ES cells.ES cells heterozygous for the disrupted allele were selected in culture medium supplemented with neomycin and ganciclovir.ES cells with targeted alleles were identified by PCR (Fig. S1A) and microinjected into C57BL/6 blastocysts to generate chimeric mice.Chimeric males were mated with C57Bl/6 females, and tail DNA of the offspring was tested for transmission of the targeted allele by PCR and sequencing of genomic DNA (Fig. 1C).The neomycin resistance cassette was removed by mating with Rosa26-flp recombinase knock-in mice 114 .Rab4A Q72L heterozygotes were back-crossed onto the C57Bl/6 strain for ten generations, as earlier described 115 .Heterozygotes were bred and wildtype (WT), heterozygote, and homozygote mice with floxed Rab4A Q72L alleles were tracked by PCR genotyping (Fig. 1D).Homozygote Rab4A Q72L mice were mated with heterozygote CD4 Cre transgenic mice to generate offspring lacking Rab4A expression in T cells (Fig. 1E).Expression of Rab4A was absent in CD4 + and CD8 + T cells of Rab4A Q72L -KO mice.
Rab4A Q72L mice have constitutive activation of Rab4A due to the elimination of GTPase activity that locks the Rab4A in a GTP-bound or active state.The mutation is ubiquitously expressed throughout the mice.The Rab4A Q72L -KO mice are the crossbreed of the floxed Rab4A Q72L mice with CD4 Cre mice where the Cre expression is dependent on the expression of CD4, which confers deletion of floxed alleles at the CD4 + CD8 + double-positive state in the thymus 38 .These results in the deletion of Rab4A in T cells of Rab4A Q72L/CD4Cre mice, termed Rab4A Q72L -KO mice (Table S1).
Animal experimentation has been approved by the Committee on the Human Use of Animals in accordance with NIH Guide for the Care and Use of Laboratory Animals.Fatal Plus (390 mg/ml pentobarbital sodium, 0.01 mg/ml propylene glycol, 0.29 mg/ml ethyl alcohol, 0.2 mg/ml benzyl alcohol preservative) was provided by the Upstate Department of Laboratory Animal Resources.Tenfold diluted Fatal Plus (100 μl per 20 g mouse) was used prior to perfusion with 2% paraformaldehyde and 1% glutaraldehyde in PBS for histological studies of the spinal cord.Cervical dislocation was used for metabolic studies to be performed in vitro.This method of euthanasia is necessary to prevent CO 2 inhalation-induced respiratory acidosis or pentobarbital-induced metabolic acidosis 116 .CO 2 or Fatal Plus administration would result in acidosis that could compromise the assessment of oxidative stress which is associated intracellular acidosis 115 .CO 2 inhalation was used for euthanasia of moribund animals.

Flow cytometry of human peripheral blood lymphocytes (PBL)
We examined unstimulated cells and cells stimulated with CD3/CD28 for 16 h 15 .T-cell subsets were analyzed by staining with antibodies to CD4, CD8, CD25, CD27, CD197, CD98, CD45RA, CD45, and CD62L.For detection of mTOR activity, cells were permeabilized with Cytofix/ CytopermPlus (eBiosciences) and stained with AlexaFluor-488conjugated antibody to pS6RP (Cell Signaling; Beverly, MA; Cat.No. 4851).Each patient's cells were freshly isolated, stained and analyzed in parallel with a matched control.Mean channel fluorescence intensity (MFI) values of patient samples were normalized to controls set at 1.0 for each analysis and expressed as fold changes.Frequencies of cell populations were compared as absolute values.Relative fluorescence intensity (RFI) was calculated by comparison of MFI values of patients' cells to healthy participants' cells, which were analyzed in parallel and normalized to 1.0.
Treatment of mice with rapamycin (Rapa) and N-acetylcysteine (NAC) Female lupus-prone mice were treated with rapamycin (Biotica, Cambridge, UK), solvent control (carboxymethylcellulose (CMC, Millipore Sigma Cat No C5013), or NAC (Spectrum Chemical Company, New Brunswick, NJ) for 12 weeks beginning at 27 ± 1.4 weeks of age.Fresh rapamycin solution was prepared daily by making a 10 mg/ml stock solution in dimethyl sulfoxide (DMSO, Millipore Sigma Cat No D2650) and making final solution in phosphate-buffered saline (PBS) with 0.2% CMC warmed to 37 °C and mixed thoroughly by vortexing.Rapamycin solution is an emulsion that precipitates out of solution when cooled, so the solution was prepared immediately and kept at 37 °C before injection.3 mg/kg rapamycin was administered in CMC solvent intraperitoneally (ip) in the left lower quadrant of the abdomen three times weekly, while 10 g/l of NAC was provided in drinking water.Control mice were treated ip three times weekly with 0.2% CMC solvent control alone.Age-matched female B6.TC and B6.TC/Rab4A Q72L -KO mice were also treated with rapamycin or solvent control.
Disease monitoring in spontaneously lupus-prone B6.TC mice Development of nephritis was monthly monitored by measurement of proteinuria from >4 weeks of age through >50 weeks of age.Urine was collected from the bottom of cages after housing mice in individual cages without bedding for 4 hours.Urine protein was measured by Bradford protein assay (Bio-Rad, catalog #500-006) using 5 μl of urine.In parallel, serum was collected by submandibular bleeding for measurement anti-nuclear auto-antibodies (ANA) and antiphospholipid autoantibodies (aPL).To assess for potential treatment toxicities, mice were weighed weekly.

Renal pathology
Kidney tissues were fixed in 10% formalin.Samples were paraffinembedded, sectioned, and stained with Periodic Acid-Schiff (PAS) and hematoxylin dyes.Histology was assessed by scoring for glomerulonephritis (GN), glomerulosclerosis (GS), interstitial nephritis (IN) on a 0-4 scale, as well as determining the percentage of sclerotic and crescentic glomeruli 13,117 .Slides were scored independently by an expert pathologist (MH) blinded to genotype and treatment groups.

Clinical evaluation of EAE
Individual animals were observed daily and clinical scores were assessed on a 0-5 scale as follows: decreased tail tone: 0.5.limp tail: 1, limp tail and hind limb weakness: 1.5, waddling gait with limp tail (ataxia): 2 ataxia with partial limb paralysis (legs slip through cage top): 2,5, ataxia with full hind limb paralysis: 3 hind limb paralysis, and forelimb weakness: 3.5, full paralysis of hind limbs and partial paralysis of forelimbs: 4, moribund: 4.5, and death: 5 119 .The animal protocol was approved by the Collaborative Institutional Training Initiative (CITI) Animal Care and Use Working Group.Data are reported as the mean clinical score ± SEM for all animals in a particular group.Mice were age and sex-matched for all experiments.Significance of clinical score of experimental over control mice was assessed by the Student's t test.

Histologic evaluation of EAE
Mice were anesthetized with 3 mg of nembutal, harvested for lymph nodes and spleen, and sacrificed by total body perfusion intracardially through the left ventricle with 25 ml of 10% formalin in 1x PBS.Brain and spinal cord were dissected out and fixed in 10% formalin.1-2 mm thick transverse serial segments were taken from the cerebral hemispheres, cerebellum/brainstem and spinal cord cervical, thoracic, lumbar and sacral regions for embedding in paraffin.Paraffin sections were stained with hematoxylin/eosin and analyzed at ×400 and ×1000 magnification for the presence of inflammatory lesions with infiltrating mononuclear cells in the meninges and parenchyma and for the presence of demyelination 120 .

Analysis of ANA and aPL by ELISA
For measurement of ANA, nuclear chromatin extract was prepared from chicken red blood cells 121 , which were pelleted at 1500 × g for 10 min and washed twice with Buffer A (80 mM NaCl, 20 mM EDTA, and 20 mM Tris-HCl pH7.5).Erythrocytes were then resuspended in buffer A with 1.5% Triton X, mixed, then placed on ice for 10 minutes and repeated once more after centrifugation at 1500 × g for 10 min.Erythrocytes were then resuspended in buffer A plus 0.25 M sucrose and pelleted at 1000 × g for 10 min.The pellet was washed twice with buffer A. The final pellet was resuspended in 10 mL of 10 mM EDTA pH 8.0 and sonicated using a Fisher Model 100 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) at 10 Watts for 60 s, cooled on ice for 1 min the repeated three more times.Protein concentration was estimated at OD 280 , with 1.0 OD 280 equal to 1.42 mg of protein.For ELISA assay, flat-bottom 96-well polystyrene plates were coated with nuclear extract (50 μg/well), cardiolipin (100 ng/well, Sigma cat.no.c1649), beta-2 glycoprotein I (β 2 GPI or apolipoprotein H, 100 pg/well, R&D Systems Cat.No. 6575-AH-050), phosphatidylethanolamine (PE, 100 ng/well; Sigma cat.no.P7693), or phosphatidylserine (PS, 100 ng/ well; Sigma cat.no.P7769) in 0.01 M NaHCO3 (pH 9.55) overnight 13,122 .After coating, plates were washed six times with 0.1% Tween-20 in PBS (Tween-20/PBS), the plates were blocked for 1 h at room temp in 10% normal goat serum in Tween-20/PBS.Antibodies were measured in 1 µl of mouse serum which was diluted 100-fold in PBS with 10% normal goat serum and 0.1% Tween-20 (Tween-20/PBS) and incubated on antigen-coated ELISA plates at room temperature for 1 h.Then, plates were washed six times with 0.1% Tween-20/PBS, and incubated with 2000-fold diluted, HRP-conjugated secondary goat antibody directed against mouse IgG (heavy and light chain) from Jackson Immuno Research Laboratories s (Cat.no.115-035-146).After washing six times with 0.1% Tween-20/PBS, plates were developed with 3,3′,5,5′-tetramethylbenzidine (TMD, Alpha Diagnostic International, Cat No 5210) and optical density (OD) was read at 450 nm and 630 nm using a Biotek Synergy II plate reader equipped with Gen5 software.Absorbances of 630 nm were subtracted from the 450 nm measurement for background reduction.In each experiment negative and positive control sera from B6 and MRL/lpr mice were used as negative and positive controls, as earlier described 13,111 .

Isolation of splenocyte subsets
Single-cell suspension was made by crushing a freshly isolated spleen through a 70-µm mesh filter (Corning Cat 431751) in a sterile Petri dish containing 10 mL of complete RPMI 1640 culture medium with 10% of fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, 10 µg/mL amphotericin B, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM β-mercapto-ethanol, and 10 mM HEPES.The suspension was washed once in 10 ml of culture medium and stored on ice until all mice were harvested to be processed together.The cell suspensions were spun down at 300 × g for 8 min at 4 o C. The supernatant were removed, and the pelleted cells were resuspended in 5 mL of ACK (150 mM NH 4 Cl, 10 mM KHCO 3, 0.1 mM EDTA, pH 7.4) lysis buffer for 5 min, after which the solution was diluted with 5 mL of complete media and spun down at 300 × G for 8 min at 4 o C. The resulting pellet was resuspended in isolation buffer (Ca 2+ and Mg 2+ free PBS supplemented with 2% fetal bovine serum and 2 mM EDTA, pH 7.4) and processed for flow cytometry or further isolation of splenocyte subsets.Cell isolations were carried out in the following sequence: i) positive CD4 isolation (ThermoFisher Scientific Cat 11461D), ii) positive CD8 positive isolation (ThermoFisher Cat No 11462D), iii) B cell negative isolation (ThermoFisher Cat No11422D).

Cell culture
Using tissue culture plates pre-coated overnight at 4 °C with 5 µg/ml of anti-CD3 (BioLegend Cat 100253), CD4 + and CD8 + T cells were cultured in complete medium containing 0.5 µg/ml soluble anti-CD28 (BioLegend Cat 102121) at 37 o C. B cells were stimulated with 10 µg/ml of lipopolysaccharide (LPS, Sigma Cat No L2630).Splenocytes were cultured with kynurenine (KYN) at or below concentrations previously employed to activate mTOR 21 .

Complete blood counts (CBC)
Blood was collected from the submandibular vein.100 µl blood was transferred into Eppendorf tubes containing 2 µl of 0.5 M EGTA for anticoagulation.The blood was stored on ice, and 20 µl of each blood sample was analyzed within 1 hour using an automated Hemavet 950 instrument (Drew Scientific, Miami Lakes, FL).

Flow cytometry
Surface receptors on live cells were stained with fluorochromeconjugated antibodies described in Table S2.Parallel detection of mitochondrial mass and potential was carried out by incubating surface-stained cells with fluorescent molecular probes (see below).Intracellular antigens, such as mTOR substrates pS6RP and pAkt, and transcription factors FoxP3 and Helios, were detected following permeabilization and fixation using FixPerm kit (eBioscience Cat Nos 00-5123-43, 005223-56, 008333-56).In agreement with a recent study 123 , we have noted far greater sensitivity and accuracy and less variability of flow cytometry as compared to western blot detection of mTORC1 and mTORC2 activities 13,15,[100][101][102] .Intracellular cytokine production was assessed following 3 h stimulation with phorbol 12-myristate 13-acetate (PMA, 5 ng/ml; Sigma, cat.no.P-8139) and ionomycin (500 ng/ml; Sigma, cat.no.I-0634) in the presence of brefeldin A (10 ng/ml, Sigma cat no.B6542).Subsequently, cells were i) stained with antibodies directed to surface antigens; ii) fixed in 1% paraformaldehyde; iii) permeabilized with the eBioscience FixPerm kit; and iv) stained with antibodies directed to cytokines (Table S2).

Receptor recycling
Freshly isolated splenocytes were stained with fluorochromeconjugated antibodies directed to surface receptors CD71 6,27 , CD38 59,60 , CD68 61,62 , CD98 63,64 , SERT 65,66 , and CD152 which are regulated through endocytic recycling 67,68 , as well as CD3, CD4, and CD8.Freshly stained cells were kept at 4 o C and used reference point for flow cytometry Unstained splenocytes were stimulated with 100 nM PDBu (Millipore Sigma, Cat.No. P1269) at 37 o C for 1 h to initiate receptor internalization 27 .PDBu was removed by washing three times in 1 ml of ice-cold PBS.To inhibit de novo protein synthesis, cycloheximide (50 µg/ml, Sigma-Aldrich cat no O1810) was added to the cells, and they were then incubated at 4 o C for 10 min.An aliquot was kept on ice, and the rest of the cells were incubated at 37 o C to allow recycling for 30 min, 60 min, and 120 min.Subsequently, the cells were stained for the expression of surface antigens 4 o C for 30 min and analyzed by flow cytometry.
Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨ m ) and mitochondrial mass ∆Ψ m was estimated by staining for 15 min at 37 o C in the dark with 1 µM tetramethylrhodamine methyl ester (TMRM, ThermoFisher Cat No T668, excitation: 543 nm, emission: 567 nm recorded in FL-2) and 20 nm 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 , Thermo-Fisher Cat No D273, excitation: 488 nm, emission: 525 nm recorded in FL-1).Co-treatment with a protonophore, 5 µM carbonyl cyanide m-chlorophenylhydrazone (mClCCP, Sigma-Aldrich Cat No 215911) for 15 min at 37 o C resulted in decreased TMRM and DiOC 6 fluorescence and served as a positive control for disruption of ∆Ψm 124 .Mitochondrial mass was monitored by staining with 100 nM Mito-Tracker Green-FM (MTG, ThermoFisher Cat No M7514, excitation: 490 nm, emission: 516 nm recorded in FL-1) or 100 nM MitoTracker Deep Red (MTDR, ThermoFisher Cat No M22426, MTDR, excitation: 644 nm, emission: 665 nm).Samples were analyzed using a Becton Dickinson LSRII flow cytometer equipped with 20 mW solid-state Ng-YAG (emission at 355 nm), 20 mW argon (emission at 488 nm), 10 mW diode-pumped solid-state yellow-green (emission at 535 nm), and 16 mW helium-neon lasers (emission at 634 nm).Data were analyzed with Flow Jo software (Version 10, TreeStar Corporation, Ashland, OR).Dead cells and debris were excluded from the analysis by electronic gating of forward (FSC) and side scatter (SSC) measurements.Each measurement was carried out on ≥10,000 cells.In each experiment, freshly isolated cells from control and lupus-prone mice were analyzed in parallel.Detailed protocols for assessment of mitochondrial dysfunction in SLE have been earlier described 125 .

Measurement of mitochondrial electron transport chain (ETC) activity and glycolysis in live cells
After 3 days of CD3/CD28 stimulation, CD4 + T cells and CD8 + T cells were seeded at 4 × 10 5 cells/well in 40 μl volume of either lymphocyte glycolysis medium (Seahorse XF Base Medium (Catalog No. 102353-100, Seahorse Bioscience and 2 mM glutamine) or mitochondrial stress test medium (Seahorse XF Base Medium, 10 mM glucose, 2 mM glutamine,1 mM sodium pyruvate) in XF 96-well culture plates coated with 450 ng/well Cell-Tak (Catalog No. 354240, Corning).The plates were centrifuged at 300 × g for 10 s with no brake to accelerate cell adherence to the plate.The cells were then equilibrated at 37 °C in ambient atmosphere (no CO 2 ) for 30 min.After the cells had fully adhered, 135 μl of media was added to each well to bring the total volume up to 175 μl.The plates were equilibrated for another 15 min at 37 °C and then loaded onto the Seahorse XFe96 Analyzer (North Billerica, MA).For the glycolysis assay, we injected 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose.For the mitochondrial stress test, we injected 1 μM oligomycin, 0.5 μM carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP), and 500 nM rotenone/antimycin A. Measurements were performed in 3-5 replicates and their means were used as the result for each experiment 126 .

Confocal immunofluorescence microscopy
For labeling and tracking of mTOR to organelles, Jurkat cell lines were incubated at 37 °C with 5 µM Lysotracker Red (catalog no.L-7528; Invitrogen) for 30 min in complete RPMI 1640 medium.Cells were washed twice and resuspended in RPMI 1640 medium followed by pipetting onto poly-L-lysine-coated (0.1 mg/ml poly-L-lysine; Sigma-Aldrich Cat No A-005-M) coverslips for 10 min at room temperature.The cells were fixed in a 4% paraformaldehyde-PBS solution for 15 min and permeabilized with a 0.1% saponin/1% FBS/HBSS mixture for 30 min.Normal goat serum (10%) was used to block the cells followed by mTOR primary antibody (catalog no.2983 S; Cell signaling) staining.Anti-mTOR antibody was directly conjugated with Alexa Flour 405 using Zenon Alexa Fluor 405 kit (ThermoFisher Cat No. Z25313).Primary antibody conjugated with Alexa Flour 405 was directly applied to the permeabilized cells for 45 min.Cells were washed twice with permeabilization buffer and twice with HBSS, mounted on slides and visualized with a Zeiss LSM 780 inverted laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 40×/1.3Plan-NeoFluar oil-immersion objective at 0.45 μm z-step intervals with lateral pixel dimensions of 0.22 μm.Images were acquired with transmission photomultiplier tube detector.Signal intensity gain was calibrated on cells that were unstained.Sequential scanning was used to record GFP (excitation: 395 nm, emission: 509 nm), mTOR-Alexa647 (excitation: 650 nm, emission: 670 nm), and LTR (excitation: 577 nm, emission: 590 nm); the RGB images were converted to 8-bit grayscales and pseudo-colored in green, blue, and red, respectively.Captured z-series were imported and analyzed using Image J. Mean intensity values were taken in each channels for each pixel, as earlier described 127,128 .Manders' overlap coefficient was obtained using JACOP plugin (available at http://rsb.info.nih.gov/ij/plugins/track/jacop.html).Colocalized signal between mTOR and LTR was quantified and divided by the mean value of the corresponding channel to determine colocalization ratio 129 .Colocalization between mTOR and LTR was assessed using an in-house developed macro.Briefly, the FIJI macro converted the red and green channel to binary images followed by calculating the total number of overlapping pixels that could not be spatially resolved between the two channels in each z-slice for the entire z-series.Sub-resolution beads were imaged with the same acquisition parameters and used to collect z-series to validate the FIJImacro 130 .

Metabolome analysis by LC-MS/MS
3 × 10 6 million cells were washed in 1 ml of PBS, pelleted at 1500 rcf in an Eppendorf centrifuge at 4 o C, and resuspended in 100 µl of 80% methanol (−80 °C) and 10 µl of 0.3 mM 5-thio-glucose, used as internal standard to allow correction for sample recovery.After freezing at −80 °C and thawing once, the sample was centrifuged at 13,000 × g for 30 min at 4 °C, and 100 µl of supernatant was saved.A 2nd aliquot of 100 µl of 80% methanol (−80 °C) was added to the pellet, the sample was vortexed, centrifuged at 13,000 × g for 30 min at 4 °C, and the 2nd 100 µl of supernatant was saved.The two 100-µl supernatants were combined, dried in a SpeedVac (Savant AS160, Farmingdale, NY), and stored −80 °C until analysis.Each sample was resuspended in 20 μl of LC/MS grade water, and 10 μl per sample was injected into a Thermo Scientific Vanquish HPLC coupled to a Thermo Scientific Q Exactive hybrid quadrupole-orbitrap MS.The metabolites were separated using a hydrophilic interaction liquid-chromatography (HILIC) method on a Waters Xbridge BEH Amide column (3.5 µm, 2.1 × 100 mm, P/N: 186004860) kept at 30 °C during the analysis.Mobile phase component A was 10 mM ammonium acetate and 7.5 mM ammonium hydroxide in water with 3 % (v/v) acetonitrile (pH 9.0) while mobile phase component B was 100% acetonitrile.The 25-min-long gradient was as follows: 0 min, 85% B; 1.5 min, 85% B; 5.5 min, 35% B; 14.5 min, 35% B; 15.0 min, 85 % B; 25.0 min, 85% B. The mobile phase flow rate was the following: 0 minutes, 0.150 ml/min; 10.0 minutes, 0.150 ml/min; 10.5 minutes, 0.300 ml/min; 14.5 minutes, 0.300 ml/min; 15.0 minutes, 0.150 ml/min, 25.0 minutes, 0.150 ml/min.The sample injection volume was 10 µL.The Thermo Scientific Q Exactive MS was operated in polarity switching mode throughout the acquisition run to maximize metabolite coverage.The heated electrospray ionization (HESI) probe parameters were the following in both polarity modes: sheath gas flow rate 30, aux gas flow rate 10, sweep gas flow rate 0, spray voltage 3.60 kV, aux gas heater temp 120 °C, S-lens RF level 55, ion transfer capillary temp 320 °C.In positive mode, the instrument acquired Full Scan spectra with a m/z range of 61-915, while in negative mode, the Full Scan mass range was m/z 70-920.The resolution of the scans was 70000, the AGC target was 3e6, while maximum IT was 200 ms.Mass spectroscopy data were collected with the following softwares: Thermo Q Exactive Tune version 2.9, Thermo TraceFinder version 4.1, and Thermo Scientific Xcalibur version 4.1.We used a quantitative polar metabolomics profiling platform with selected reaction monitoring (SRM) that covers all major metabolic pathways to confirm the identity of targeted metabolites.The platform uses hydrophilic interaction liquid chromatography with positive/negative ion switching to analyze ∼ 500 metabolites from a single 25-min acquisition run with a 3-ms dwell time and a 1.55-s duty cycle time (Supplementary Table S4).
Metabolite steady-state, pathway, and flux analyses Quantitative enrichment of detected metabolites was utilized for pathway analysis employing the web-based MetaboAnalyst 5.0 software 132 .Samples from compared mice were matched for age and gender and were injected in the same LC-MS/MS run.The signal stability was assured by normalizing the controls between runs to the sum of all signals between separate runs using Metaboanalyst.The enrichment analysis was based on global analysis of covariance (Ancova).A Google-map style interactive visualization system was utilized for data exploration and creation of a 3-level graphical output: metabolome view, pathway view, and compound view.The "metabolome view" shows all metabolic pathways arranged according to the scores from enrichment analysis (y axis: −log p) and from topology analysis (x axis: impact: number of detected metabolites with significant p value).The pathway topology analysis used two well-established node centrality measures to estimate node importance: degree centrality and betweenness centrality.Degree centrality depends on the number of links connected to a given node.For directed pathway graphs, there are two types of degrees: in-degree for links came from other nodes, and out-degree for links initiated from the current node.Here, we only considered the out-degree for node importance measure.Upstream nodes are considered to have regulatory roles for the downstream nodes, and not vice versa.The betweenness centrality measures the number of shortest paths going through the node.Since metabolic networks are directed, we used relative-betweenness centrality for a metabolite importance measure based on metabolite topology weighed by relative-betweenness centrality 132 .The degree centrality measures focus more on local connectivity, while the betweenness centrality measures focus more on global network topology.The node importance values calculated from centrality measures were further normalized by the sum of the importance of the pathway.Therefore, the total/maximum importance of each pathway reflects the importance measure of each metabolite node that is actually the percentage relative to the total pathway importance, and the pathway impact value is the cumulative percentage from the matched metabolite nodes.The altered compounds have been grouped and presented together for each pathway.
Metabolite concentrations were evaluated for their ability to discriminate between WT, Rab4AQ72L and Rab4AQ72L-KO mice by partial least squares-discriminant analysis (PLS-DA) 133 .Contribution of individual metabolites to PLS-DA was assessed by variable importance in projection (VIP) and coefficient scores.Individual compounds were also compared between B6 and lupus-prone mice by 2-way ANOVA paired and Tukey's correction for multiple comparisons using Prism Software Version 10 (GraphPad, San Diego, CA).
For metabolic flux analyses, stable isotope-labeled compounds [1,2-13 C]-glucose (CLM-504-0.5),[U- 13  No GLC-032).Following CD3/CD28 stimulation for 72 h, the culture medium was replaced with glucose-free medium supplemented with 13 C or 2 H stable isotope labeled glucose or glutamine-free medium replaced with 13 C-labeled glutamine for 30 min.Metabolites of stable isotope labeled compounds were tracked through the mitochondrial TCA cycle, glycolysis, and the PPP (Fig. S13) 126 .% enrichment of labeled metabolites was compared to unlabeled compounds.

Fig. 1 |
Fig. 1 | Rab4A activation promotes ANA, aPL, proteinuria, and GN in female B6.TC mice.Effect of Rab4A activation and T-cell-specific inactivation were examined on antinuclear autoantibody (ANA; A), β2-glycoprotein I or apolipoprotein H (Apo-H; B), and anti-cardiolipin autoantibody (ACLA; C) production and proteinuria (D) in age-matched, 20-to 29-week-old, male and female mice homozygous for constitutively active Rab4A Q72L or lacking Rab4A in T cells Rab4A Q72L -KO, respectively.E Effect of Rab4A on the development of GN, GS, and % hyalinosis in female mouse sets at ~50 weeks of age.Scale bars are embedded into each representative microscopic image.Dot plots present individual mice.F Effect of Rab4A on the development of GN, GS, and % hyalinosis in male mouse sets at ~50 weeks of age.Kidneys were scored by an experienced renal pathologist blinded to mouse genotypes.Scale bars are embedded into each representative microscopic image.Dot plots present individual mice.2-way ANOVA and Sidak's post-hoc test p values are displayed for multiple comparisons of Rab4 WT, Rab4A Q72L , and Rab4A Q72L -KO mice within B6 control and B6.TC SLE strains.Overall 2-way ANOVA p values are shown in the header of each figure panel, while Sidak's post-hoc test p values < 0.05 over brackets reflect comparison between experimental groups.

Fig. 6 |
Fig. 6 | Contrasting effects of KYN on activation of mTORC1 and mTORC2, expression of CD98 and relative abundance of primary CD4 + and CD8 mouse T + cells.Splenocytes from four female B6 mice were stimulated with 1 mM KYN alone or together with CD3/CD28 for 72 h in vitro, as indicated for each panel.Phenotyping for expression of CD3, CD19, CD4, CD8, and CD98 was performed by flow cytometry.A Measurement of mitochondrial mass and ROS production by MTG and HE fluorescence, respectively.Left panels show representative flow cytometry dot plots, while right panels show GraphPad charts of cumulative analyses.Brackets represent p values < 0.05 by comparison using two-tailed paired t test.The number of mice was n = 4 in each experimental group.Charts show mean ± SEM for each experimental group.B Effect of KYN on the expression of CD98 and the prevalence of CD4 + and CD8 + T cells with and without concurrent CD3/CD28 co-stimulation.Representative flow cytometry histograms and dot plots and mean ± SE four independent experiments are shown.Brackets show p values < 0.05 using 3-way ANOVA and Sidak's post-hoc tests to correct for multiple comparisons.CD98MFI three-way ANOVA p = 0.0264; Sidak's post-hoc test p values corrected for multiple comparisons: KYN vs control unstimulated CD4 + T cells p < 0.0001, KYN vs control unstimulated CD8 + T cells p < 0.0001, KYN vs control CD3CD28-stimulated CD4 + T cells p < 0.0001, KYN vs control CD3CD28-stimulated CD8 + T cells p < 0.0001, CD3CD28-stimulated vs unstimulated CD4 + T cells p = 0.0009, CD3CD28-stimulated vs unstimulated CD8 + T cells p < 0.0001, control CD3CD28-stimulated CD4 + vs control CD3CD28-stimulated CD8 + T cells p = 0.0003, KYN-treated CD3CD28-stimulated CD4 + vs KYN-treated CD3CD28 -stimulated CD8 + T cells p < 0.0001.Two-way ANOVA of CD3CD28stimulated control and KYN-treated CD4 + and CD8 + T cells p = 0.0100.%CD98 + cells three-way ANOVA p = 0.6602; Sidak's post-hoc test p values corrected for multiple comparisons: KYN vs control unstimulated CD4 + T cells p = 0.0048, KYN-treated unstimulated CD4 + T cells vs KYN-treated unstimulated CD8 + T cells